Dec 17, 2016 - metal ion receptor. The fluorescence signal decreased to 46% upon Fe ion binding to the metal receptor of calcein. However, this probe cannot ...
Serial Review Journal JCBN the 1880-5086 0912-0009 Kyoto, Serial 10.3164/jcbn.16-70 JCBN16-70 Society Review Japan of Clinical for FreeBiochemistry Research and Nutrition Japan Advances inRadical Chemical Tools for Exploring Oxidative Stress Guest Editor: Hidehiko Nakagawa
Chemical tools for detecting Fe ions Tasuku Hirayama* and Hideko Nagasawa Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, 1254 Daigakunishi, Gifu 5011196, Japan (Received 10 August, 2016; Accepted 13 September, 2016; Published online 17 December, 2016) ??
Owing to itsopen electrochemical with of inter Creative stricted vided Copyright This 2017 the is use, original an Commons distribution, ©distinctive 2017 work access JCBN Attribution isarticle and properly reproduction distributed License, cited. properties under which in anythe permits medium, terms unreprothe convertible multiple oxidation states, iron plays a significant role in various physiologically important functions such as respiration, oxygen transport, energy production, and enzymatic reactions. This redox activity can also potentially produce cellular damage and death, and numerous diseases are related to iron overload resulting from the dysfunction of the iron regulatory system. In this case, “free iron” or “labile iron,” which refers to iron ion weakly bound or not bound to proteins, causes aberrant produc tion of reactive oxygen species. With the aim of elucidating the variation of labile iron involved in pathological processes, some chemical tools that can qualitatively and/or quantitatively monitor iron have been utilized to investigate the distribution, accumula tion, and flux of biological iron species. Since iron ions show unique reactivity depending on its redox state, i.e., Fe2+ or Fe3+ (or transiently higher oxidative states), methods for the separate detection of iron species with different redox states are preferred to understand its physiological and pathological roles more in detail. The scope of this review article covers from classical chro mogenic to newly emerging chemical tools for the detection of Fe ions. In particular, chemical tools applicable to biological studies will be presented. Key Words:
iron, fluorescenct probe, chromogenic probe, imaging
A
AIntroduction n adult human accumulates ca. 3–4 g of iron in his body, this amount being prominently high as compared to other transition metals. Iron is involved in a considerable number of physiologically important processes such as respiration, oxygen transport, signal transduction, and enzymatic reactions in a biological system(1–3) relying on its ability to activate molecular oxygen. Activation of oxygen means generation of reactive oxygen species (ROS).(4) Under physiologically normal conditions, the rate between production and consumption/detoxification of ROS is strictly controlled to exploit the beneficial aspects of ROS. Dysfunction of the iron homeostasis process could seriously affect the balanced ROS metabolism leading to the aberrant production of ROS, thus, resulting in cell damage and death.(4) In particular, the Fenton reaction has been known as one of the most harmful reactions in a biological system where Fe2+ acts as an activator to generate highly toxic hydroxyl radicals (or highly reactive chemical species) that can induce DNA damage and peroxidation of lipids.(5–7) Thus, an excess of iron has been reported to be highly relevant to serious diseases, such as cancer,(8,9) and neurodegenerative diseases including Alzheimer and Parkinson, where deposits of iron are often observed in patients suffering these diseases.(10–12) Moreover, some indexes relevant to biological iron such as the total serum iron concentration, total iron binding capacity, and the unsaturated iron binding capacity have been
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clinically used for diagnosing hemochromatosis and anemia.(13) Since iron has multiple oxidation states, Fe2+ or Fe3+ (or transiently higher oxidative states), and shows unique reactivity depending on the redox state, utilizing analytical methods for separate detection of iron at different redox states is essential to deeply understand the physiological and pathological roles of this metal ion. This review article focuses on not only classical but also emerging chemical tools for detecting Fe ions. In particular, chemical tools applicable to biological studies will be presented. Chromogenic Chelators for Colorimetric Methods Metal complexes with chelators sometimes show characteristic intense colors (i.e., absorption in the visible region) attributed to their metal to ligand charge transfer (MLCT) and/or ligand to metal charge transfer (LMCT) band. The color changes are generally dependent on the metal concentration, thereby enabling accurate quantification of metal ions on the basis of molar absorptivity of the complexes. In this context, colorimetric methods using chromogenic chelators have been widely used to quantitatively determine the concentration of iron ions. Although tissues and cells need to be homogenized prior to measurements, colorimetric methods enable easy and precise quantification with conventional instruments such as UV–vis spectrometers and microplate readers. In this section, we present several chromogenic chelators for the selective detection and quantification of iron ions. Bathophenanthroline (BP) and bathophenanthroline disulfonic acid disodium salt (BPS) (Fig. 1a). 4,7-Diphenyl-
1,10-phenanthroline, known as BP,(14) has been developed as an indicator of Fe2+. BP showed an improved molar absorptivity of 22,400 cm−1 M−1 at 533 nm upon formation of a 3:1 complex with Fe2+ as compared to its parent compound 1,10-phananthroline (11,100 cm−1 M−1). In the first report on colorimetric methods with BP, total iron in aqueous samples was reduced by hydroxylamine followed by extraction with water-insoluble alcohols such as isoamyl and n-hexyl alcohols. The optical density (OD) or absorbance at 533 nm of the ferrous complex extracted into the alcohol fraction was measured and compared with standard solutions to determine the exact concentration of Fe2+ ions. The detection range with a cuvette of 1 cm path length was 0.1–1 ppm (1.8– 18 μM). The BP method had been employed as standard method to determine the concentration of serum iron by the International Committee for Standardization in Haematology (ICSH), although it is not recommended at present.(15,16) BPS was produced by the introduction of two hydrophilic sulfonates on BP. Since it is the water-soluble analogue of BP, *To whom correspondence should be addressed. Email: hirayamat@gifupu.ac.jp
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Fig. 1.
Chemical tools for the colorimetric detection of Fe ions. (a) BP and BPS, (b) Ferrozine, (c) FereneS, and (d) NitrosoPSAP.
it enables direct application to aqueous sample without the need of extraction processes.(17) Introduction of two hydrophilic sulfonates on BP enhances water-solubility. Sulfonation and its application in aqueous samples did not alter its molar absorptivity (22,140 cm−1 M−1), color (λabs = 535 nm), and sensitivity characteristics. It is noted that the presence of excess copper ion may interfere with quantification of iron via formation of a 2:1 complex with BP or BPS (λabs = 425 nm) in the presence of reductants such as ascorbate and hydroxylamine. Ferrozine and FereneS (Fig. 1b and c). 3-(Pyridin-2-yl)5,6-bis(4-sulfophenyl)-1,2,4-triazine disodium salt, known as Ferrozine,(18) is a highly water-soluble chromogenic chelator of Fe2+. Ferrozine has one pyridyl group and two phenylsulfonate groups on a 1,2,4-triazine core. The two sulfonate groups provide high water-solubility characteristics and allow utilization in aqueous samples. The chelating motif of the pyridyl-tetrazine core forms a 3:1 complex with Fe2+ having an intense color (absorption centered at 563 nm) with high molar absorptivity (27,900 cm−1 M−1). The presence of five equivalents of cobalt (Co2+)
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and copper (Cu+) introduces 5% and 15% error, respectively. 3(Pyridin-2-yl)-5,6-bis(5-sulfofuran-2-yl)-1,2,4-triazine disodium salt, known as Ferene-S, is an analogue of Ferrozine where the 4-sulfonylphenyl groups are substituted with 5-sulfonylfuryl groups.(19) Ferene-S presents absorption maximum at 593 nm and a molar absorption of 34,500 cm−1 M−1, thereby improving sensitivity up to ca. 1.3-fold compared to Ferrozine. The selectivity and limitations are similar to those of Ferrozine. Quantitative iron detection kits based on Ferrozine or Ferene-S are now commercially available and recommended as standard methods of iron quantification by ICSH and the Clinical and Laboratory Standards Institute (CLSI).(15,20) NitrosoPSAP (Fig. 1d). A series of nitrosophenol derivatives had been known to show absorption maxima in the near infrared region upon binding to Fe2+ ion. 2-Nitroso-5-(N-propylN-sulfopropylamino)phenol, known as Nitroso-PSAP, shows higher water-solubility and selectivity to Fe2+ ion as compared to other transition metal ions.(21,22) The colorimetric response to Fe2+ ion via formation of complex is highly specific (i.e., unique
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Fig. 2. Absorbance spectrum of ironchelating chromogenic chelators complexed with metal ions (cited from ref. (23): Clin Chim Acta, 2014; 437: 129–135). Black: Fe2+, red: Cu2+, green: Co2+, blue: Ni2+. (a) BP, (b) Ferrozine, (c) Ferene, and (d) NitrosoPSAP.
absorption wavelength at 756 nm) with high molar absorptivity (45,000 cm−1 M−1). This response can be clearly differentiated from those showed by the complexes with other metal ions such as Co2+ (493 nm), Cu2+ (430 nm), and Ni2+ (391 nm). This large separation of the absorption band enables selective and accurate quantification of Fe2+ ion in aqueous samples in the presence of other metal ions. Thus, Nitroso-PSAP shows a low error of only 7% in the presence of Cu2+. Recently, Ito et al.(23) reported a wellestablished assay system of non-transferrin-bound iron (NTBI) by exploiting the Nitroso-PSAP system. In this report, they compared the spectral changes of Nitroso-PSAP, BP, Ferene-S, and Ferrozine when forming complexes with Fe2+, Cu2+, Co2+, and Ni2+ to decide the best chromogenic chelator for their iron detection system (Fig. 2). All the colorimetric methods described above are wellestablished and accurate methods for quantification of Fe2+ in aqueous samples without the need of expensive instruments such as atomic absorption (AAS) and inductive-coupled plasma mass spectrometers (ICP-MS) and radioisotopes (59Fe). However, these colorimetric methods require treatment of the sample with reductant, surfactant, and/or acid agents to prepare a homogenous solution, thereby making it hard to apply to living samples. Fluorescent Probes Unlike colorimetry, fluorescent probes are capable of monitoring metal ions in cuvette and in living cells/animals. Fluorescent probes are categorized into three types depending on the mode of signal change: turn-on, turn-off, and ratiometric probes. Turn-on and turn-off probes show increase and decrease in fluorescence signal against the targets (iron), respectively. Ratio-
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metric probes provide shifts in emission or excitation wavelength and readout of ratio between two intensities. Generally, transition metal ions having unoccupied d-orbitals exhibit strong fluorescence-quenching effects due to their paramagnetic effect.(24) Therefore, both Fe3+ and Fe2+ act as quenchers of fluorescence, and the majority of early fluorescent probes for iron ions exhibit turn-off responses resulting from the quenching effect of the metal ions. Ratiometric probes provide shifts in emission or excitation wavelength and ratio readout between two intensities. In this section, we present the progress in the development of fluorescent probes for Fe3+ and Fe2+ ranging from early turn-off probes to emerging turn-on and ratiometric probes. Early fluorescent probes with turnoff response. The first fluorescent probe of Fe ion is calcein. Breuer et al.(25,26) found that calcein and its acetoxymethyl ester (calcein-AM) (Fig. 3a) can work as turn-off fluorescent probes of Fe ion. These probes consist of fluorescein as a fluorophore and two iminodiacetate arms as a metal ion receptor. The fluorescence signal decreased to 46% upon Fe ion binding to the metal receptor of calcein. However, this probe cannot distinguish between Fe2+ and Fe3+ since Fe2+ is readily oxidized to Fe3+ upon binding to calcein. These authors explored cell-based fluorometry with calcein-AM to monitor the uptake of Fe ion by K562 cell and succeeded in observing attenuation of fluorescence signal as a result of metal binding to calcein and subsequent recovery of the signal by loading salycylaldehyde-isonicotinoyl-hydrazone (SIH)(27) or BIP (2,2'bipyridyl),(25) which remove Fe3+ and/or Fe2+ from calcein. These experiments represent the first success to trace intracellular iron flux by fluorometry.(28) PhenGreen-SK (PGSK), which is composed of 2',7'-dichlorofluorecein tethered with 1,10-phenanthroline, was subsequently proposed as a Fe2+-selective chelator. PGSK
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Fig. 3.
Classical turnoff fluorescent probes of iron ions. (a) Calcein and (b) PhenGreen SK (PGSK).
forms a 3:1 complex with Fe2+ and exhibits turn-off response (Fig. 3b). Rauen and Petrat performed fluorescence imaging experiments with PGSK and revealed that “chelatable iron ions” or “labile irons” are present in living hepatocytes. The binding affinity of PGSK to Fe2+ is much stronger than calcein and too strong to dissociate Fe2+ from the chelating moiety of PGSK by other iron chelator, and therefore, calcein has been favored to study cytoplasmic iron pools.(29) The endogenous labile iron ions were found to exist as Fe2+ rather than Fe3+ by using a Fe2+selective chelator, 2,2'-bipyridyl.(30) Petrat et al.(31) reported RPA {rahodamine B-[(1,10-phenanthroline-5-yl)aminocarbonyl]benzyl ester}, an analogue of PGSK, which can localize the mitocondria and monitor the mitochondrial flux of Fe2+ (Fig. 4). Although RPA is a turn-off type fluorescent probe, they succeeded in detecting accumulative iron in mitochondria caused by inhibition of heme synthesis. Remarkably, this study indicates that Cu2+ can also induce quenching of RPA. These authors also reported utilization of RDA and PIRO (Fig. 4a), new probes bearing 2,2'-bipyridyl and dipyridylmethylamine, respectively, which were designed to monitor mitochondrial Fe2+ as analogues of RPA.(32) RDA showed improved affinity to Fe2+ as compared to RPA, while that of PIRO was not intense enough to detect endogenous Fe2+ in mitochondria. Calcein, calcein-AM, and PGSK are commercially available at present and widely used to monitor labile iron in living systems by fluorescence microscopy and fluorometry. For the use of such turn-off type fluorescent probes, it is necessary to distinguish positive from false-positive responses owing to photo-bleaching and leakage of the probe from cells. Moreover, both probes show similar turn-off responses to other metal ions such as Ni2+, Cu2+, and Co2+, among others. A large number of probes for Fe3+ have been reported, although the majority of them show turn-off type response. Several probes have iron-binding ligands derived from siderophores, which are natural iron-chelating compounds secreted by plants and bacteria to uptake insoluble Fe3+ from soil. FL-DFO(33) and NBD-DFO(34) (Fig. 4b) have desferrioxamine (DFO) motifs for iron binding. DFO is a highly selective and strong siderophore to Fe3+, which
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makes these probes highly selective and sensitive to this ion. 3-Hydroxy-1,2-dimethylpyridin-4-one, known as deferiprone (DFP), is clinically used as an iron-exporting agent(35) and utilized to develop several turn-off probes. A series of Fe3+ probes consisting of coumarin fluorophore conjugated with a DFP motif have been reported. Among them, the cell membrane permeable analogues (CP645 and CP800, Fig. 4c), have quenching efficiencies for Fe3+ of 93% and 66%, respectively.(36) These two probes showed significant quenching characteristics in live cell applications using hepatocyte cells. Turn-off responses were also observed against Fe2+ because of acceleration of oxidation (from Fe2+ to Fe3+) upon chelation, thereby indicating that this type of probes with DFP as a metal receptor enable to measure total labile iron concentrations. The same group reported a derivative of CP800 to monitor lysosomal labile iron (Fig. 4c).(37) The main drawback of the siderophore-inspired probes described above is their turn-off readout, which causes low spatial resolution while imaging and false positive derived from photo bleaching and leakage of the probes, as noted above. Turnon fluorescent probes of Fe3+. Unlike turn-off fluorescent probes, turn-on fluorescent probes for aqueous Fe3+ are still limited. Hua and Wang(38) reported the first turn-on probe of Fe3+ that can work in aqueous media (Fig. 5, Fe3+-1). This probe consists of two cyclic polyether groups attached on a 7aminocoumarin moiety. This probe showed a 15-fold increase in fluorescence at 412 nm (λex = 336 nm), with the response being highly selective to Fe3+. The affinity (Ka) toward Fe3+ was calculated to be 1.5 × 105 M−1 in the form of 1:1 complex. This probe also showed a minor increase in fluorescence (2.5-fold) upon Cu2+ with a Ka of 2.4 × 103 M−1, which can be considered negligible compared to its response against Fe3+. This probe requires UV light (336 nm) for excitation. BODIPY-based turn-on fluorescent probe for Fe3+ (Fig. 5, Fe3+2) was reported by Bricks et al.(39) They utilized a macrocycle (3-aza-10-oxa-1,7-dithiacyclododecane) as a Fe3+-binding receptor, which was attached on the meso-position of the BODIPY dye. The probe showed over 500-fold increase in its fluorescence intensity at 512 nm (λex = 470 nm) upon addition of Fe3+ or Cu+
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Fig. 4. Turnoff fluorescent probes applicable to live cell imaging. (a) Mitochondriatargeted probes. (b) Siderophorebased probes. (c) Deferiprone based probes.
in acetonitrile, this increase being 15-fold in weakly acidic aqueous solution (pH 5.1 or 5.8) or non-buffered water. Unlike the aqueous system, the response was not selective in acetonitrile. The stoichiometry between the probe and Fe3+ was 1:1, with Ka = 1.6 × 104, 1.0 × 104, 1.3 × 104 M−1 in MOPS buffer (pH 5.1), tris HCl buffer (pH 5.8), and water, respectively. This probe can be excited by visible wavelength for detection, although no biological application was demonstrated. Controlling the equilibrium between spirocycle and openquinoid structures of the rhodamine derivative has been established (summarized in Fig. 5, right column) as a reliable strategy to design fluorescent probes for Fe3+.(40) Xiang and Tong(41) (Fe3+3) first employed rhodamine spirolactam to develop a turn-on fluorescent probe by tethering two rhodamine B spirolactams with a 3-aza-pentylene linker. The spirocyclic structure of rhodamine B was dominant in aqueous media within a neutral pH range (5.0–9.0), resulting in no visible absorption and fluorescence. Upon binding to Fe3+, the spirolactam opened via coordination of oxygen or nitrogen atoms to Fe3+. Probe Fe3+-3 responded selectively to Fe3+ in neutral aqueous buffer showing a fluorescence enhancement rate of 114-fold at 572 nm (λex = 510 nm). The fluorescence response was reversible by addition of EDTA.
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However, the affinity of the probe was not high enough for cellular applications (Ka = 3.1 × 103 M−1). Once the design strategy using rhodamine spirolactam was established, a number of fluorescent probes having different chelating motifs emerged on the basis of similar strategies. Since a good review covering all these probes has been recently published elsewhere,(40) herein we focused on examples that are applicable to aqueous media (
